Use of N2O in the treatment of post-ischemic brain cell deterioration

ABSTRACT

A medicinal composition and method to treat, minimize, or prevent post-ischemic brain cell deterioration that comprises nitrous oxide or a nitrous oxide donor. The method comprises administering the medicinal composition prior to or subsequent to a stroke.

[0001] The invention relates to the use of nitrous oxide (N₂O) or of anN₂O donor for producing all or part of a medicinal product intended totreat or prevent post-ischemic brain cell deterioration, in particulardeterioration subsequent to a stroke, especially all or part of aninhalable gaseous medicinal product, in humans or animals.

[0002] In cerebral ischemia subsequent to a stroke, and in strokes ingeneral, a functional alteration of many neurotransmission systems isusually noted from a neurochemical point of view, in particular anincrease in the release of glutamate, the excitotoxicity andcontribution of which to neuronal death are known, as recalled byDirnagl et al., Trends Neurosci, 22: 391, 1999.

[0003] Moreover, from a functional point of view, in the case of globalischemia in the rat, an increase is observed in locomotor activity, inparticular described by Wang and Corbett, Brain Res., 533: 78, 1990;Baldwin et al., Neurodegeneration 2: 139, 1993, the development of whichis generally attributed to an alteration in cognitive functions ofspatial recognition rather than to an alteration in sensory-motorfunctions.

[0004] As a result, a potential therapeutic role for ionotropic andmetabotropic glutamergic receptor antagonists have been suspected, inparticular by Chazot, Curr Opin Invest Drugs 1: 370, 2000; Drian et al.,Neurochem Int 38: 509, 2001.

[0005] It also appears that the deleterious effects of known cerebralischemias appear to involve localized ischemias which are thought to becaused by glutamergic excitotoxicity.

[0006] In fact, the therapeutic potential of glutamergic receptorantagonists is often put forward in the treatment of neuropathologies ofexcitotoxic origin, in particular cerebral ischemia, as described byDirnagl et al., Trends Neurosci 22: 391, 1999, and productive disorders,as described by Benes, Brain Res. Review 31: 251, 2000.

[0007] However, the physiology of glutamergic receptors is complex andit appears that the high affinity antagonists may also exhibitneurotoxic properties, according to Burns et al., Psychopharmacology115: 516, 1994.

[0008] Thus, a potential therapeutic advantage of low affinityantagonists, in particular for NMDA, has recently been proposed byPalmer and Widzowski, Amino acids 19: 151, 2000.

[0009] Moreover, document WO-A-02/09731 is also known, which relates tothe use of CO, optionally supplemented with another gas, for treatinginflammations of the upper respiratory tracts or of the bronchi. Thatdocument therefore targets the treatment of pathologies such as asthma,cystic fibrosis, pneumopathies or the like.

[0010] In addition, document EP-A-861672 teaches a method of treatmentwhich can be used in emergency situations by administering variousgases. However, this does not relate to post-ischemic brain celldegradations subsequent to strokes.

[0011] Finally, document FR-A-2812545 teaches a combination of gas andactive product intended to treat or prevent pain. The active product isan analgesic, an anti-inflammatory, an anti-pyretic or the like.

[0012] To date, no effective medical product therefore exists forpreventing or treating, at least partially, post-ischemic brain celldegradation subsequent to strokes.

[0013] The present invention falls within this context, and aims toprovide all or part of a medicinal product which can be used forpreventing, decreasing or treating any post-ischemic brain celldeterioration, in particular subsequent to a stroke, in humans oranimals.

[0014] The invention therefore relates to the use of nitrous oxide (N₂O)or of an N₂O donor for producing all or part of a medicinal productintended to treat, minimize or prevent post-ischemic brain celldeterioration.

[0015] Depending on the case, the use of the invention may comprise oneor more of the following technical characteristics:

[0016] all or part of the gaseous medicinal product is in inhalableform;

[0017] the post-ischemic brain deterioration results in or is subsequentto a stroke;

[0018] the nitrous oxide (N₂O) or the nitrous oxide donor is in gaseousform or is included in a gas or a mixture of gases;

[0019] the medicinal product contains an effective proportion of nitrousoxide (N₂O) or of N₂O donor;

[0020] the medicinal product also contains xenon or a xenon donor, thexenon or the xenon donor being in gaseous form or being included in agas or a mixture of gases;

[0021] the medicinal product contains an effective proportion of xenonor of a xenon donor;

[0022] the medicinal product also contains at least one other gaseouscompound chosen from oxygen, nitrogen or argon, preferably nitrogen andoxygen;

[0023] the medicinal product contains an amount which is less than 60%by volume of xenon or of xenon donor, preferably less than or equal to50% by volume;

[0024] the medicinal product contains an amount ranging up toapproximately 80% by volume of N₂O or of N₂O donor, preferably up to 75%of N₂O;

[0025] the medicinal product contains from 19 to 25% by volume of oxygenand, optionally, of nitrogen.

[0026] The invention therefore also relates to an inhalable medicinalproduct with neuroprotective action in the brain, containing aneffective amount of nitrous oxide (N₂O) or of a donor of such acompound, in particular intended to treat, minimize or preventpost-ischemic brain cell deterioration.

[0027] According to the case, the medicinal product of the invention maycomprise one or more of the following technical characteristics:

[0028] it contains an amount ranging up to 80% by volume of gaseous N₂O;

[0029] it contains an effective amount of nitrous oxide (N₂O) or of adonor of such a compound;

[0030] it contains an effective amount of xenon or of a donor of such acompound;

[0031] it contains an amount which is less than 60% by volume of xenon;

[0032] it also contains from 19 to 25% by volume of oxygen and,optionally, of nitrogen.

[0033] The idea on which the present invention is based is to takeadvantage of the NMDA receptor antagonist properties of gaseous N₂O,optionally supplemented with xenon, for their neuroprotective nature, inprevention or treatment of post-ischemic pathologies subsequent tostrokes.

[0034] In fact, recent studies, carried out in vitro, have shown thatN₂O, and also xenon, can potentially behave like low-affinityantagonists of glutamergic receptors for N-methyl-D-aspartate, NMDA(Franks et al., Nature 396: 324, 1998; Jevtovic-Todorovic et al., NatureMed. 4: 460, 199; Yamakura and Harris, Anesthesiology, 20008).

[0035] Based on these observations, experiments were carried out in thecontext of the present invention, with the aim of determining theneuroprotective effects of N₂O and of xenon, on neuronal death inducedby transient cerebral ischemia in rats.

[0036] In order to demonstrate the beneficial effect of administeringN₂O or xenon on brain cells subsequent to cerebral ischemia, adultSprague-Dawley rats weighing 350 g were subjected to the followingexperimental protocol.

[0037] On day 1, focal ischemia was induced in each of the rats bymiddle cerebral artery occlusion (MCAO), for a period of 1 h 30 minutes.

[0038] The transient focal cerebral ischemia by MCAO is obtainedconventionally by introducing a flexible nylon thread 1, representeddiagrammatically in FIG. 1 (length 6.5 mm, diameter 180 μm), a portion 2of the proximal end of which has a diameter greater than that of thethread (length 3 mm, diameter 380 μm), into the vascular system of therat, as far as the region of the ipsilateral hemisphere so as to causean embolism therein, i.e. an ischemia.

[0039] Next, the rats are reperfused for 10 to 20 minutes, and are thenmade to inhale several mixtures of gases, namely:

[0040] mixture No. 1: air (control)

[0041] mixture No. 2: N₂O (75% vol), the remainder being oxygen (25%)

[0042] mixture No. 3: xenon (50% vol), the remainder being oxygen (20 to25%) and nitrogen (30 to 25%), respectively

[0043] mixture No. 4: xenon (75% vol), the remainder being oxygen (25%).

[0044] On day 2, i.e. 24 hours after reperfusion, the rats are killed,the brains are recovered and frozen, and thin sections 40 μm thick arecut and then stained with cresyl violet, as shown in FIG. 5.

[0045] The volume of neuronal death is calculated, from the sectionsobtained after staining, in a conventional manner using an appropriate,commercially available conventional program.

[0046] In fact, as shown diagrammatically in FIG. 2, the cerebralischemia engenders, in general, in 24 hours, an infarction in the regionwhich has been subjected to ischemia (penumbra), leading to neuronaldeath in the brain cells present in a considerable portion of thisregion.

[0047] The results obtained during these measurements have been recordedin FIGS. 3a to 3d, which make it possible to visualize the post-cerebralischemia neuroprotective effect of mixtures No. 2 to 4 above, incomparison with mixture No. 1 (air) which serves as a control.

[0048] Thus, FIG. 3a clearly shows that inhalation by the rats ofnitrous oxide (N₂O) or of xenon (Xe) subsequent to an ischemia makes itpossible to considerably reduce the total volume of infarction, since adecrease in this volume of approximately 50% can be achieved in the caseof inhalation of mixtures No. 2 and No. 3 instead of air (mixture No. 1acting as control), and of approximately 30% when mixture No. 4 isinhaled.

[0049] In this respect, it will also be noted that inhalation of 50% byvolume of xenon (mixture No. 3) is more effective than inhalation of ahigher dose of xenon, namely 75% (mixture No. 4), which implies that themost effective dose appears to be closer to 50% than to 75% with regardto xenon.

[0050] FIGS. 3b to 3d confirm the results of FIG. 3a, since they make itpossible to observe that inhalation of xenon or of N₂O makes it possibleto decrease, respectively, the post-ischemic volume of corticalinfarction (FIG. 3b), the post-ischemic volume of striatal infarction(FIG. 3c) and the post-ischemic volume of oedema (FIG. 3d), compared toinhalation of air (control=mixture No. 1).

[0051] Based on this observation, complementary examinations werecarried out in order to determine the neurotoxic effects of the xenonand of the nitrous oxide (N₂O), at various amounts, compared to air, onbrain receptors of the NMDA type.

[0052] The results of these examinations are reported in FIG. 4, whichclearly shows that the administration of xenon or of nitrous oxideengenders a smaller volume (in mn³) of deteriorated NMDA receptors thanthe control (air), this being with the nitrous oxide given at a dose of50% or 75% by volume (remainder=25% of O₂) and the xenon given at a doseof 50% or 75% (remainder=mixture of 25% of O₂+25% of N₂, or,respectively, 25% of O₂).

[0053] However, a neurotoxic effect which is variable according to thedose administered thus emerges, leading to the observations that N₂O at75% and xenon at 50% by volume are more neuroprotective than N₂O at adose of 50% and xenon at a dose of 75%.

[0054] In other words, these data confirm that administration byinhalation of N₂O at a dose of 75% by volume (or less) or of xenon at adose of 50% by volume (or less) engenders a neuroprotective action withrespect to cerebral ischemia and other similar excitotoxic diseases.

[0055] The inhalable medicinal product according to the invention ispackaged in pressurized gas containers, such as gas bottles, and isdispensed to the patient via an appropriate system for administeringgas, equipped with a breathing mask, a tracheal catheter, or the like.

[0056] Consequently, in the context of the invention, the use of gaseousnitrous oxide (N₂O) will be preferred to that of xenon, for producing amedicinal product intended to treat, minimize or prevent post-ischemicbrain cell deteriorations, such as strokes.

1-15: (canceled). 16: A medicinal composition to treat, minimize, orprevent post-ischemic brain cell deterioration that comprises at leastone component selected from the group consisting of: a) nitrous oxide,and b) a nitrous oxide donor. 17: The medicinal composition according toclaim 16, wherein said composition further comprises at least onecomponent selected from the group consisting of: a) xenon, and b) axenon donor. 18: The medicinal composition according to claim 16,wherein said composition further comprises at least one gaseouscomponent selected from the group consisting of: a) oxygen, b) nitrogen,and c) argon. 19: The medicinal composition according to claim 18,wherein said gaseous component comprises: a) oxygen, and b) nitrogen.20: The medicinal composition according to claim 16, wherein at leastpart of said nitrous oxide or nitrous oxide donor is in gaseous form.21: The medicinal composition according to claim 16, wherein saidcomposition is inhalable. 22: The medicinal composition according toclaim 17, wherein said xenon or xenon donor is in gaseous form. 23: Themedicinal composition according to claim 16, wherein said compositioncomprises an effective proportion of said nitrous oxide or nitrous oxidedonor. 24: The medicinal composition according to claim 16, wherein saidnitrous oxide or nitrous oxide donor comprises about 80% by volume ofthe total composition. 25: The medical composition according to claim24, wherein said nitrous oxide or nitrous oxide donor comprises about75%. 26: The medicinal composition according to claim 17, wherein saidxenon or xenon donor comprises less than about 60% by volume of thetotal composition. 27: The medicinal composition according to claim 26,wherein said xenon or xenon donor is equal to or less than about 50%.28: The medicinal composition according to claim 18, wherein said oxygenis in the range of from about 19% to about 25% by volume of the totalcomposition. 29: The medicinal composition according to claim 19,wherein said oxygen is in the range of from about 19% to about 25% byvolume of the total composition. 30: The medicinal composition accordingto claim 16, wherein said nitrous oxide is inhalable. 31: The medicinalcomposition according to claim 16, wherein said composition is placed ina pressurized gas container. 32: The medicinal composition according toclaim 31, wherein said container is a gas bottle. 33: A method fortreating, minimizing, or preventing post-ischemic brain celldeterioration comprising: i) administering a medicinal compositioncomprising nitrous oxide or nitrous oxide donor. 34: The methodaccording to claim 33, wherein said medicinal composition is inhalable.35: The method according to claim 33, wherein said method furthercomprises: ii) adding xenon or a xenon donor to the composition. 36: Themethod according to claim 33, wherein said method further comprisesadding at least one component selected from the group consisting of: a)oxygen, b) nitrogen, and c) argon. 37: The method according to claim 36,wherein said method comprises adding: a) oxygen, and b) nitrogen. 38:The method according to claim 35, wherein said xenon or xenon donor isin gaseous form. 39: The method according to claim 33, wherein saidnitrous oxide or nitrous oxide donor is in gaseous form. 40: The methodaccording to claim 33, wherein said method further comprisesadministering a therapeutically—effective amount of said nitrous oxideor nitrous oxide donor. 41: A method for treating, minimizing, orpreventing post-ischemic brain cell deterioration comprising: i)administering a medicinal composition comprising nitrous oxide ornitrous oxide donor, and ii) providing neuroprotective action in thebrain.